Tuberculosis is the leading cause of death from a single infection agent killing more than 3 million people per year worldwide. In the year 1998, the estimated numbers of Tuberculosis cases in India were 2,078,076 among which 935,134 cases were likely to be infectious (WHO report, 1998) and it has been nearly 40 years since the introduction of a novel compound for the treatment of tuberculosis. 
The consequence of tuberculosis infection is clearly an outcome of the continuous interplay between the pathogen and the host immune defense. In most instances, the infected individual mounts an effective immune response that culminates in granuloma formation around the infective foci and cessation of disease progression. Clinical studies suggest that the bacilli within these granulomas are not killed but instead remain dormant (Grange, 1992; Stead, 1967; Stead et al., 1968). This is termed a latent infection. Natural TB infection without treatment can lead to latent infection, which can last a lifetime and approximately 10% of latent infections reactivate, resulting in active disease months to years after the initial infection (Stead et al., 1968). The large number of latently infected individuals presents a major impediment to reducing the incidence of tuberculosis and the rate of M. tuberculosis transmission. The adaptation of the M. tuberculosis during the spectrum of infection and disease, including prolonged survival within granulomas, is likely implemented through precise genetic pathways that are modulated by specific physiological and environmental conditions within host tissues. There is an urgent need to understand these pathways in order to devise novel and more directed strategies for the prevention, control and treatment of tuberculosis.
Rate limiting steps in metabolic pathways that are unique to prokaryotes, such as cell wall biosynthesis and DNA replication, have been traditional foci of anti-infectives. Their poor activity against slow growing and non-replicating bacteria is thought to be an important reason why currently used regimens take so long to eradicate infection (Parrish et al., 1998) and often fail to eradicate at all. The rapid explosion in our understanding of the M. tuberculosis genome and its metabolic pathways has provided an unprecedented opportunity to design drugs to novel metabolic targets.
Discovery of bacterial genes that play key roles in persistence has opened the way for the identification of molecules in dormant bacteria that can be targeted by new classes of drugs. Bacterial two-component systems belong to this class of novel targets (Barrett and Hoch, 1998).
Two-component signal transduction systems are the most basic form of sensory systems which involve direct response to environmental signals like stress (O2 tension, pH), nutritional deficiencies (chemical compounds, biological analogs, ions), exposure to chemicals, toxins and changes in osmolarity, etc. to name a few. The two-component system consists of a basic sensory system comprising a transmembrane sensor histidine kinase which senses the environmental stimulus and undergoes autophosphorylation at a conserved histidine residue in an ATP-dependent manner (FIG. 1). The phosphorylated histidine sensor kinase then transfers the phosphoryl moiety to a conserved aspartate residue of the response regulator protein, the other half of the two-component system, which is predicted to possess DNA binding ability due to the presence of a conserved helix-turn-helix motif present in it. The phosphorylation status of the response regulator protein can alter its DNA binding potential/ability and consequently switch on or off the expression of the genes under its regulatory control. (Parkinson and Kofoid, 1992; Stock et al., 2000).
The complete genome sequencing of M. tuberculosis H37Rv, a virulent strain of M. tuberculosis capable of causing tuberculosis, revealed the presence of genes potentially coding for eleven complete two-component systems and seven orphan sensor kinase and response regulator proteins (Cole et al., 1998). Out of these systems only four systems have been characterized at biochemical level including DevR-DevS (described herein), DevR-Rv2027c (described herein), SenX3-RegX3 (Himpens et al., 2000) and TrcS-TrcR (Haydel et al., 1999). The DevR-DevS (Rv3133c-Rv3132c) two-component system has been suggested to play a regulatory role during oxygen limitation on account of its hypoxia-responsive pattern of gene expression (Tyagi, J S, DST report, October 2001; Boon et al., 2001, Sherman et al., 2001). Hypoxia is postulated to constitute a trigger for the onset of mycobacterial dormancy within granulomas. The DevR-DevS system is therefore a candidate for regulating dormancy of the tubercle bacili. On the basis of the experimental evidence which have been amassed about the role of this two component system in the virulence of Mycobacterium tuberculosis, DevR-DevS system is an appropriate choice as a target for the action of antibiotics to inhibit its putative function and subsequently eradicate/kill the tubercle bacilli or to prevent the bacilli from going into dormant stage. It can also be used to supplement conventional therapies and antibiotics which are not effective against the dormant bacilli. For screening the inhibitors against this two-component system, the phosphorylation pathways of DevR-DevS and DevR-Rv2027c (orphan sensor kinase highly homologous to DevS) were established at the biochemical level.
Drugs which inhibit two-component systems are expected to be specific for bacteria and not for humans since the latter are not reported to contain two-component systems. The involvement of the DevR-DevS two-component system, and particularly of response regulator, DevR in virulence provides an attractive target for chemotherapeutic agents directed tubercle bacilli (Kapur, V., Ph.D. Thesis, 2001; Tyagi and Kapur, PCT/IN02/00022). Isocitrate lyase (McKinney et al., 2000) a methyl transferase pcaA (Glickman et al., 2000), and a two-component system Rv0981-0982 (Zahrt and Deretic, 2001) have also been identified as crucial to the persistent state of the tubercle bacilli. These genes together with some other recently discovered targets like resuscitation factors (Mukamolova et al., 1998) which include 8 kDa secreted protein and proteins homologous to 16 kDa protein of Micrococcus luteus which act as resuscitation factor (Mukamolova et al., 1998), provide new avenues for improved therapy of tuberculosis (Barry et al., 2000).
A number of genes that have been earlier implicated in dormancy/persistence have been characterized at various levels. The three-dimensional crystal structures of some of the proteins involved have been characterized, including isocitrate lyase and antigen 85B (Armitige et al., 2000). Based on their 3-D structural lattices, rational drug design is currently underway in several laboratories. However, functional dissection of a mycobacterial regulatory network cascade involving a two-component system in general, and DevR-DevS system in particular, has not been accomplished yet. In this context DevR-DevS forms a potent target for attacking bacteria residing within granulomas. Oxygen limitation and hypoxia has been recently shown to upregulate the synthesis of DevR-DevS two-component system in M. tuberculosis, M. bovis BCG and M. smegmatis in an in vitro dormancy model (Tyagi, J S, DST report, October 2001; Sherman et al., 2001; Boon et al., 2001 and Mayuri et al., 2002). This finding has crucial implications for M. tuberculosis since hypoxia often is thought to be a characteristic environmental property of granulomas and a likely trigger for the initiation and/or maintenance of dormancy response. Since DevR is upregulated under hypoxia, modulation of gene activity by DevR is likely to be achieved through its putative DNA binding activity as is known to occur in other bacterial two-component system response regulators (Dziejman and Mekalanos, 1995). A number of genes induced during hypoxia and implicated in persistence therefore may be under regulation of DevR-DevS two-component system.
Besides candidate genes involved in persistence and/or dormancy response of M. tuberculosis as targets for anti-tubercular therapy, a number of other gene targets have also been suggested as intervention targets due to their critical role in M. tuberculosis survival and pathogenesis (Zhang and Amzel, 2002) including, (i) transcription factors like stationary-phase sigma factor SigB (Doukhan et al., 1995), SigF (DeMaio et al., 1996), global expression regulating sigma factor, SigE and SigH (Manganelli et al., 2001; Raman et al., 2001), (ii) virulence associated factors like RpoV (Collins et al., 1995), catalase-peroxidase, KatG (Wilson et al., 1995), complex lipid phthiocerol dimycocerosate (PDIM) and its transporter mmpL7 (Cox et al., 1999), (iii) cell-wall synthesis regulating genes like phosphatidylinositol synthetase (Jackson et al., 2000), fatty-acid synthase type II (Kremer et al., 1999) and (iv) two-component systems like MtrA which have been shown to be essential for M. tuberculosis survival (Zahrt and Deretic, 2000); PhoP/PhoQ and Rv0981/Rv0982 which regulates virulence (Perez et al., 2001; Zahrt and Deretic, 2001). In addition to these defined approaches there are many targets and/or genes in the M. tuberculosis which are under investigation on the basis of their homology to other known microbial genes involved in pathogenesis (Zhang and Amzel, 2002). Many other genes which are now targeted for anti-tubercular therapy are essentially the one whose 3-D structure has been elucidated and hence they can be used effectively in structure-based rational drug-design approach, for example arylamine N-acetyltransferases—NAT (Upton et al., 2001), iron-dependent regulator—IdeR (Pohl et al., 1999), antigen 85 complex (Ronning et al., 2000; Anderson et al., 2001), iron-dependent superoxide dismutase—SOD (Zhang et al., 1991; Cooper et al., 1995) and many others. Despite such a huge number of genes targeted for anti-tubercular therapy, no new interventions have been reported to date.
Chemical modification of existing classes of antibacterial agents continues to be a fruitful approach to the design of new antibiotics, as evidenced by the discovery of glycopeptides effective against vancomycin-resistant enterococci and ketolides with activity against streptomycin-resistant S. pneumoniae (Baltch et al., 1998; Denis et al., 1999). Such an approach was utilized for devising new anti-tubercular drugs like rifampicin derivatives like rifapentine etc. But, incremental changes in the structure of poorly efficacious antibacterials, however, are likely to afford analogues with a limited life span due to established resistance to the parent drug (Macielag and Goldschmidt, 2000). This further necessitates the development of completely novel and newer drug targets and therapeutic modalities effective against tuberculosis.
A scientifically appealing but largely unexplored approach was to inhibit the function or expression of virulence factors and/or regulatory elements like two-component systems. The two-component regulatory systems have received increasing attention both a novel antibacterial drug targets and as potential sites of action for virulence inhibitors (Barrett and Hoch, 1998; Wallis, 1999).
The two-component systems or sensor kinase or response regulator proteins as targets for new drugs or compounds have been utilized in recent past (Macielag and Goldschmidt, 2000). Reports from independent laboratories, including pharmaceutical companies, have provided various compounds with proven potential to inhibit the phosphorylation of sensor histidine kinases in particular in in vitro assays. However, to date such a strategy has not been attempted for developing antimycobacterial agents.
The vast majority of two-component system inhibitors described block the autophosphorylation of histidine sensor kinase component. However, the precise mode of action of such inhibitors has generally remained obscure due to lack of published enzyme kinetics data, making it difficult to develop a useful pharmacophore model. Moreover, most of the inhibitors in the literature were discovered before the structures of the sensor kinase catalytic domains were published.
Most of the sensor kinase inhibitors identified by broad-spectrum corporate library screening have turned out to be highly hydrophobic compounds which include, for example, isothiazolones (Ulijasz et al., 1999), fatty acid derivatives (Strauch et al., 1992), imidazolium salts (Roychoudhary et al., 1993), tyramine structural motifs like cyclohexene derivatives (Urbanski et al., 1997) and benoxazines (Frechette et al., 1997). Most of the identified compounds have shown efficacy in in vitro assays wherein they have shown the potential to inhibit the phosphorylation assays, which was used as the screening reaction or assay.
There are three main reasons for identifying and developing new antitubercular drugs: (i) to improve current treatment by shortening the total duration of treatment and/or by providing for more widely spaced intermittent treatment, (ii) to improve the treatment of MDR-TB, and (iii) to provide for more effective treatment of latent tuberculosis infection. These reasons summarize all the major drawbacks in existing antitubercular therapy and indicate the needs to be addressed.
Drug resistant forms of tuberculosis pose a serious threat to the successful outcome of a tuberculosis control programme in a community. Although many highly effective drugs such as isoniazid, rifampicin, ethambutol and pyrazinamide are available, poor compliance due in part to long treatment schedules (6 months is the standard duration) leads to high rates of treatment failure. There is an urgent need to implement the DOTS (Directly observed short course) treatment schedule to reduce the incidence and spread of tuberculosis in general and drug resistant forms in particular. Though conventional drug regimens comprising administration of rifampicin-isoniazid-pyrazinamide is very effective, the minimum inhibitory concentration (MIC) for all the three drugs is very close to the maximum serum concentration, which is limited by toxicity, resulting in poor therapeutic index for each. Serum concentration of these drugs may oscillate between levels above and below the MIC over the course of daily administration. This phenomenon, coupled with poor patient compliance over the course of this lengthy chemotherapy, has been proposed to be linked directly to the emergence of drug resistance (Mitchison, 1998). This ineffectiveness of current therapies is therefore directly responsible for both the very long duration of therapy and the emergence of resistance to drugs. Certain fourth generation quinolones including gatifloxacin and levamofloxacin etc. have shown certain degree of efficacy against many—a clinical isolates of mycobacteria including M. tuberculosis under in vitro conditions, but these drugs are also marred by the same intrinsic drawbacks like their limited efficacy under in vivo conditions, severe side-effects, high cost and rapid emergence of resistant isolates.
Therefore there is a pressing need to introduce new drugs that would be effective against resistant forms of tuberculosis and also reduce the duration and cost of chemotherapy. Although many lead compounds targeted against the conventional drug targets are being tested worldwide, no new drugs for tuberculosis have been introduced in the market over the last thirty years.
In addition, there is no drug available in the market today which can be used to tackle the ever expanding problem of latent tuberculosis. All the drugs which are used for combating tuberculosis, are effective only in the active disease conditions and are not able to eradicate the latent disease. Since the DevR-DevS two-component system has been implicated in the virulence and in dormancy particularly, targeting it would also provide a handle to counter the problem of latent tuberculosis. Technically, a genetic disruption of response regulator gene, devR of the DevR-DevS and/or DevR-Rv2027c two-component system signal transduction pathways is equivalent to an attenuated strain which is rapidly cleared off from the system and fails to cause any latent tuberculosis (Kapur, V., Ph.D. Thesis, 2001; Tyagi and Kapur, PCT/IN02/00022). Consequently disruption of DevR-DevS or DevR-Rv2027c signal transduction pathways by the means of inhibitors would be equivalent to their genetic disruption and a conventional therapy alongside this would effectively eradicate tubercle bacilli without allowing it to enter a dormant stage.
The utilization of two-component systems as screens for inhibitors have provided a number of inhibitor molecules which have shown potential to inhibit the autophosphorylation reaction of sensor kinases in in vitro assay systems. But, all the identified inhibitors and compounds for bacterial two-component systems suffer from the drawback of extreme hydrophobicity. Such high hydrophobicity of these molecules makes formulation and delivery of the compounds extremely difficult. Furthermore, the compounds showed minimal bioavailability and excessive plasma protein binding, Thus, the compounds have been ineffective in standard in vivo infection models.
The benzooxazines (Frechette et al., 1997) were designed with the goal of improving the hydrophobic/hydrophilic balance and the in vivo activity of the inhibitors. Though these compounds demonstrated good antibacterial activity, they also suffered from excessive protein binding, which hampered in vivo efficacy. A series of sensor kinase inhibitors identified by Hoch et al. (1998) included the established antihelminthic compound Closantel and RWJ-49815, which turned out to be molecules leading to non-specific aggregation of sensor kinase molecules in an in vitro assay rather than causing inhibition of phosphorylation reaction (Stephenson et al., 2000). A series of bisphenol analogs identified at Parke-Davis Pharma inhibited NRII autophosphorylation and showed inhibition of functional responses mediated by the two-component system NRII/NRI etc. in whole cell assays (Domagala et al., 1998) and were bactericidal in action at higher concentrations as well. But, the mechanism of cell death was shown to be membrane-disruption leading to complete shutdown of macromolecular synthesis. The compounds are also ineffective in vivo due to excessive plasma protein binding. Thus far, all identified inhibitors of autophosphorylation of histidine sensor kinases have turned out to be hydrophobic compounds that were often difficult to formulate adequately for in vivo protection studies. Whenever tested, these compounds failed to protect in mouse models of infection probably due to excessive serum protein binding.
Halophenylisothiazolones were shown to inhibit the transfer the phosphate from the histidine protein kinase to the response regulator in a reconstituted VanS/VanR signal transduction pathway employing a membrane preparation of the VanS kinase and purified VanR (Ulijasz and Weisblum, 1999). Mechanistic studies demonstrated that the compounds inhibited the acceptor activity of VanR rather than the donor activity of VanS˜P. Since inhibition occurred at higher concentration of inhibitor (ED50=0.35 mM), this may account for the discrepancy in the reported mechanism of action. However, isothiozolone derivatives had already shown their inefficacies in in vivo studies as mentioned above.
Imidazolium compounds was reported to inhibit the binding of AlgR1 response regulator to an algD upstream region/probe at a concentration of ˜150 μM in a gel-mobility shift assay (Roychoudhary et al., 1993). The compound was claimed to be a specific inhibitor of transcription from the algD promoter as opposed to a non-specific inhibitor of DNA-protein interaction, due to lack of activity on CatR binding to the catBC box. However, as mentioned above the imidazolium derivatives and analogs clearly have a general effect on histidine protein kinase autophosphorylation as well and are quite ineffective in in vivo protection assays.
In general, most of these agents appear to suffer from poor selectivity, excessive protein binding or limited bioavailability.
As mentioned above there is an urgent need to identify novel drug targets for the development of new drugs that would be effective against tuberculosis that is resistant to treatment by drugs that are in use today. Furthermore there is a grave need for effective drugs that can target chronic/latent forms of tuberculosis in contrast to the currently administered drugs that target actively replicating bacilli.
Earlier reports from Dr. Jaya S. Tyagi's laboratory have suggested that the devR gene is more likely to be related in chronic infection process and is less relevant to the growth per se in human monocytes or in the early events of infection. It was observed that the mutant strain failed to cause severe progressive disease and pathology under the experimental conditions in guinea pigs as compared to the wild type H37Rv strain (Kapur, V., Ph.D. Thesis, February 2001, Tyagi and Kapur, PCT/IN02/00022).
Since, the DevR protein belongs to the response regulator class of regulatory proteins; it is plausible that it orchestrates the adaptation of tubercle bacilli to the hostile environment of the host. The two-component system's regulatory network functions through a phosphorylation pathway, where DevS histidine sensor kinase (HK) protein senses a environmental cue or stimulus in response to which it undergoes autophosphorylation at a conserved His395 residue. The phosphorylated DevS species then transfers the phosphoryl moiety to the DevR protein via a phosphotransfer event at a conserved Asp54 residue. A likely phosphorylation-induced change in DevR protein structure likely changes its DNA binding ability, leading to modulation in expression of genes under its control. It is known that this two-component system is responsive to hypoxia (Boon et al., 2001; Sherman et al., 2001; Mayuri et al., 2002), a key factor involved in persistence and it is also believed that this system is indeed involved in virulence of tubercle bacilli (Kapur, V., Ph.D. Thesis, February 2001; Tyagi and Kapur, PCT/IN02/00022). It thus provides a novel target for the development of drugs active against the bacilli located in the granulomas. It is believed that disabling the function of a regulatory system such as DevR-DevS and DevR-Rv2027c will lead to the inactivation of bacterial pathway(s) modulated by them in response to hypoxia or other virulence and pathogenesis-associated signals. The present invention provides the protocol and biochemical assays which can be utilized to screen for inhibitor molecules, compounds, agents or drugs against these pathways or systems which lead to functional inactivation of the system (FIG. 1).
In light of the deficiencies of the two-component system inhibitors from corporate compound libraries, alternate strategies can be employed for identification and design of inhibitors against two-component systems. A rational drug-designing and screening approach involving X-ray or NMR structure of the cytosolic domain of the sensor kinase in combination with computer applications for de novo design and screening of inhibitors have been suggested by many groups (Inouye U.S. Pat. No. 6,162,627). Furthermore, instead of screening inhibitors for phosphorylation reactions, inhibitors can be screened to the other sites in the two-component system pathway, for example, (i) dimerization domain of the sensor kinase, (ii) sensory domain to the sensor kinase, (iii) sensor kinase and response regulator interaction interface, and (iv) response regulator—DNA interaction interface, which is possible only with the detailed understanding of the catalytic activities of the respective participating proteins, which forms an essential part of this invention.
Besides using these modified selections or screening strategies, natural product or combinatorial peptide libraries can also be used as a source of novel two-component system inhibitors in high-throughput assays. Once a lead ‘candidate’ molecule is identified in high-throughput screening, it should be extendable to screening in whole cell assays also in high throughput assay format.
Though, the DevR-DevS and/or Rv2027c-DevR two-component systems offers unique regulatory systems as targets for developing novel anti-microbial/anti-bacterial/anti-tubercular compounds, the utilization of novel refolding screen and specific mutant proteins also offers a means for effectively screening potent inhibitors via the rational drug design approach using the DevR modeled structure or by using the mutant proteins in screening steps for peptide library screening setup.
The present invention involves utilization of DevR-DevS (Rv3133c-Rv3132c) and Rv2027c-DevR signal transduction pathways as targets for therapy against diseases caused by mycobacterial organisms including all forms of Mycobacterium tuberculosis and other mycobacteria possessing these two-component systems. The invention further covers the utilization of these proteins and their catalytic activities as modes for screening antibacterial, antimycobacterial, bactericidal and/or bacteriostatic drugs and/or compounds that target all or any steps of these signal transduction pathways.
Rv2027c protein is 62.5% identical to DevS protein and contains H, N, D/G1 and G2 boxes typical of histidine kinases. It was speculated that Rv2027c being an orphan sensor kinase, could be autophosphorylated and in turn participate in an phosphotransfer event with DevR protein. As described herein this hypothesis was tested and confirmed to occur in vitro.
It is hence proposed to use these phosphorylation assays or reactions to screen for lead molecules that block these phosphorylation reactions and utilize them as bactericidal/antimicrobial compounds capable of interfering with these signal transduction pathways and thereby inhibiting the expression of downstream gene targets under their control and blocking their physiological manifestations such as, for example, dormancy or latency (see FIG. 1).